Thyroid disruptions by xenobiotics have been associated with a broad spectrum of severe adverse human health effects, such as impaired brain development and metabolic syndrome. Ingestion of indoor dust and contact with industrial chemicals are two significant human exposure routes of thyroid hormone disrupting chemicals (THDCs), raising serious concerns for human health. However, it is a laborious and costly process to identify THDCs using conventional experimental methods, due to the number of chemicals in commerce and the varieties of potential disruption mechanisms. In this thesis, we are aimed at in silico identification of novel THDCs targeting transthyretin (TTR) and thyroid hormone receptor (THR) among dust contaminants and commonly used industrial chemicals. In vitro assays were used to validate the in silico prediction results. Co-crystallization and molecular dynamics (MD) simulations were applied to reveal binding modes of THDCs at the studied biological targets and to explain their intermolecular recognition. The main findings presented in this thesis are: 1. Over 144 environmental pollutants have been confirmed as TTR-binders in vitro and these cover a wide range of environmental pollutants and show distinct chemical profiles including a large group of halogenated aromatic compounds and a second group of per- and polyfluoroalkyl substances. (Paper I) 2. In total 485 organic contaminants have been reported to be detected in household dust. The developed QSAR classification model predicted 7.6% of these dust contaminants and 53.1% of their metabolites as potential TTR-binders, which emphasizes the importance of metabolic bioactivation. After in vitro validation, four novel TTR binders with IC50 ≤ 10 µM were identified, i.e. perfluoroheptanesulfonic acid, 2,4,2',4'-tetrahydroxybenzophenone (BP2), 2,4,5-trichlorophenoxyacetic acid, and 3,5,6-trichloro-2-pyridinol. (Paper II) 3. The development of a robust structure-based virtual screening (VS) protocol resulted in the prediction of 31 dust contaminants as potential binders to THRβ1 including musk compounds, PFASs, and bisphenol A derivatives. The in vitro experiments confirmed four compounds as weak binders to THRβ1, i.e. 2,4,5-trichlorophenoxyacetic acid, bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether, 2,4,2',4'-tetrahydroxybenzophenone, and 2,4-dichlorophenoxyacetic acid. (Paper III) 4. We revealed the binding conformations of perfluorooctanesulfonic acid, perfluorooctanoic acid, and BP2 in the thyroxine binding sites (TBSs) of TTR by co-crystallizing TTR with the three compounds. A VS protocol was developed based on the TTR complex structures that predicted 192 industrial chemicals as potential binders to TTR. Seven novel TTR binders were confirmed by in vitro experiments including clonixin, 2,6-dinitro-p-cresol (DNPC), triclopyr, fluroxypyr, bisphenol S, picloram, and mesotrione. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr, and their complex structures showed that the compounds bind in the TBSs as proposed by the VS protocol. In summary, 13 indoor dust contaminants and industrial chemicals were identified as THDCs using a combination of in silico and in vitro approaches. To the best of our knowledge, none of these compounds has previously been reported to bind to TTR or THR. The identifications of these THDCs improve our understanding on the structure-activity relationships of THDCs. The crystal structures of TTR-THDC complexes and the information on THDC-Target intermolecular interactions provide a better understanding on the mechanism-of-actions behind thyroid disruption. The dataset compiled and in silico methods developed serve as a basis for identification of more diverse THDCs in the future and a tool for guiding de novo design of safer replacements.

O hormônio tireoidiano (HT) participa do controle de funções essenciais do organismo. A maioriados seus efeitos é mediada pela modulação da transcrição gênica e se manifesta em um período longo o suficiente para permitir a transcrição de genes específicos. Por outro lado, são crescentes na literatura as evidências de que o HT também promove efeitos que ocorrem em um curto espaço de tempo e que se manifestam mesmo na presença de inibidores da transcrição gênica. O GLUT4 é o principal transportador de glicose presente no músculo esquelético, no cardíaco e no tecido adiposo. O seu processo de translocação e inserção na membrana plasmática resulta da ativação de vias de sinalização que ocorre a partir da interação da insulina com seus receptores de membrana. No músculo esquelético e cardíaco, uma segunda via que aciona o mecanismo de translocação do GLUT4 envolve a ativação da AMPK, processo desencadeado pela contração muscular. O presente estudo teve como objetivo avaliar: (i) no modelo in vivo (ratos Wistar), se o T3 e o T4 provocam agudamente a translocação do GLUT4 para a membrana plasmática; (ii) no modelo in vitro (células musculares L6 e adipócitos 3T3-L1), se o T3 e o T4 provocam o efeito descrito acima; e (iii) se esse efeito ocorre por ativação das vias de sinalização da insulina e/ou da contração muscular. Nossos estudos in vivo demonstram que a administração de T3 rapidamente aumentou o conteúdo de GLUT4 na fração correspondente à membrana plasmática no músculo esquelético e no tecido adiposo. No entanto, essa ação foi independente da ativação da PI3-K e da AMPK. Os estudos in vitro, mostraram que o T3 promove, rapidamente, um aumento na captação de glicose nas células L6 sem, contudo, alterar o conteúdo de GLUT4 presente na membrana. Esses resultados sugerem que essa ação do T3 ocorra devido a ativação do GLUT4 já presente na membrana ou devido a algum processo independente dessa proteína. Nossos resultados demonstram que ao lado das suas reconhecidas ações genômicas, o HT atua por mecanismos não genômicos regulando a translocação do GLUT4. Além disso, sugerem fortemente que o T3 participe, também por mecanismos não genômicos, do processo de ativação do GLUT4 já inserido na membrana. / The thyroid hormone (TH) participates in the control of essential functions of the organism. Most of its effects are mediated by modulation of gene transcription and take place over a long enough period of time to allow the transcription of specific genes. On the other hand, evidence that TH also promotes the effects that occur in a short period of time and which manifest even in the presence of inhibitors of gene transcription have been increasingly found in literature. GLUT4 is the main transporter of glucose in skeletal muscle, the heart and adipose tissue. Its translocation and insertion in the plasma membrane result from the activation of signaling pathways triggered by the interaction of insulin with membrane receptors. In skeletal muscle and the heart, a second pathway that activates the mechanism of GLUT4 translocation involves the activation of AMPK, a process triggered by muscle contraction. This study aimed at evaluating: (i) in the in vivo model (Wistar rats), if T3 and T4 acutely cause translocation of GLUT4 to the plasma membrane, (ii) in the in vitro model (L6 muscle cells and adipocytes 3T3 -L1), if T3 and T4 cause the effect described above; and (iii) whether this effect occurs by activation of the signaling pathways of insulin and/or muscle contraction. Our in vivo studies demonstrate that administration of T3 rapidly increased the amount of GLUT4 in the fraction corresponding to the plasma membrane in skeletal muscle and adipose tissue. However, this action did not depend on the activation of PI3-K and AMPK. In vitro studies showed that T3 quickly increases the glucose uptake in L6 cells, but without changing the amount of GLUT4 present in the membrane. These results suggest that this action of T3 occurs due to activation of GLUT4 already present in the membrane or due to some process which does not depend on this protein. Our results demonstrate that other than its known genomic actions, TH acts through nongenomic mechanisms regulating GLUT4 translocation. In addition, they strongly suggest that T3 participates, also through non-genomic mechanisms, in the activation process of GLUT4 already inserted in the membrane.

Dados recentes mostram que o remodelamento ósseo está sujeito ao controle do SNC, com o SNS. Para tanto, avaliamos o (CLO) e a morfologia da (LE) do fêmur de camundongos de 21 dias de idade selvagens (WT) e Knockout (KO) para a2CAR, tratados, para mimetizar o hipertiroidismo (Hiper) ou tratados para indução do hipotiroidismo (Hipo). Os animais KO eutiroideos (Eut) apresentaram uma desorganização da ZP e aumento do número de CHM da LE. Nos animais WT, o Hipo promoveu redução do CLO, desorganização da ZP e diminuição do número de CHM. O Hipo promoveu efeitos contrários na LE dos animais KO em relação aos animais WT, sendo capaz de reverter, parcialmente, a desorganização da ZP dos camundongos KO Eut, além de resultar em maior número de CHM, em relação aos animais WT Hipo. Já o Hiper levou a aumento do número de CH nos animais WT e redução nos animais KO. Os animais KO Hiper apresentaram diminuição de CHM, quando comparados aos animais WT Hiper. Esses achados reforçam as hipóteses de que o SNS regula o CLO via a2CAR e que o HT e SNS interagem para regular o CLO. / Recent data show that bone remodeling is under control of the CNS, with the SNS. Thus, we evaluated the bone growth and the morphology of the femoral EGP of 21- day old female wild-type (WT) and a2CAR-/- mice (KO), treated to mimic hyperthyroidism (Hyper), or treated for hypothyroidism (Hypo). The euthyroid KO mice had a disorganization of PZ and increase in the number of MHC of the EGP. In WT animals, Hypo promoted a significant reduction in the BLG, PZ disorganization and a decreased number of MHC. Hypo promoted opposite effects in EGP of KO compared to WT mice. Hypo was able to partially revert the PZ disorganization observed in euthyroid KO, and resulted in a greater number of MHC compared to WT Hypo. On the other hand, Hyper caused an increase in the number of HC in WT mice and a reduction in KO mice. Furthermore, Hyper KO animals showed a reduction in the number of MHC, when compared to the Hyper WT mice. These findings support the hypothesis that the SNS regulates the BLG via a2CAR and that the interaction between SNS and TH can regulate the BLG.

Sabe-se que o hormônio tireoideano (HT) é essencial para o crescimento e desenvolvimento ósseos. No entanto, os mecanismos pelos quais o HT regula esses processos são pouco entendidos. Recentemente, o sistema nervoso simpático (SNS) foi identificado como um potente regulador do metabolismo ósseo. Estudos do nosso grupo mostraram que o HT interage com o SNS para regular a massa e estrutura ósseas, e que essa interação envolve a sinalização adrenoceptora α2 (α2-AR). Também identificamos a presença de todos os subtipos de adrenoceptores α2 adrenérgicos, o α2A- , α2B- e α2C-AR, na lâmina epifiseal (LE) de camundongos. Além disso, observamos que camundongos com inativação gênica isolada do α2A-AR e α2C-AR (α2A-AR -/- e α2C-AR -/- ) apresentam LEs desorganizadas, ossos longos mais curtos, e atraso na ossificação endocondral. Estudos in vivo revelaram, que as LEs de animais α2A-AR -/- e α2C-AR -/- respondem de forma diferente (do que as LEs de animais selvagens) ao excesso e deficiência de HT, o que sugere fortemente que o HT também interage com o SNS para regular o crescimento e o desenvolvimento ósseos. Através de um sistema de cultura de órgãos de ossos longos, o presente estudo teve como objetivo investigar se o HT interage com o SNS diretamente no esqueleto, para regular o crescimento linear ósseo, e se os receptores α2 adrenérgicos estão envolvidos nessa interação. Assim sendo, avaliamos, in vitro, o crescimento linear ósseo de tíbias derivadas de embriões de camundongos (com 15 dias de vida intrauterina) selvagens (WT) e α2C- AR -/- (KO) durante 6 dias. Vimos que as tíbias KO apresentam um menor crescimento longitudinal quando comparadas às tíbias WT, e que o tratamento com 10-8 M de triiodotironina (T3) diminuiu significativamente o crescimento longitudinal das tíbias WT, o que não foi visto nas tíbias KO. Vimos, ainda, que o tratamento com UK 14,304 (UK), um agonista α2 não seletivo, induziu o crescimento longitudinal somente nas tíbias KO. A expressão de genes relacionados com a diferenciação terminal de condrócitos (Col X, IGF-1, Wnt-4 e Runx2) mostrou-se aumentada nas tíbias KO (quando comparada à expressão nas tíbias WT). O tratamento com T3, como esperado, estimulou a expressão desses genes nas tíbias WT, porém diminui a expressão nas amostras KO, chamando a atenção para a importância desses receptores na modulação das ações do T3. Observamos, ainda, que a ativação local dos α2- ARs com o UK bloqueia a expressão desses genes relacionados à diferenciação dos condrócitos, além de bloquear os efeitos positivos do T3 (UK+T3) na expressão desses genes. Esses achados mostram que os receptores α2 adrenérgicos atuam diretamente no esqueleto para controlar a diferenciação terminal dos condrócitos e, portanto, o crescimento longitudinal ósseo, além de permitirem uma interação com a via de sinalização do HT para controlar esses processos. Em conclusão, este estudo mostra que HT interage com o SNS, localmente no esqueleto, via sinalização α2 adrenérgica, para modular o crescimento linear ósseo. / It is well known that thyroid hormone (TH) is essential for normal bone growth and development. However, the mechanisms by which TH regulates these processes are poorly understood. Recently, the sympathetic nervous system (SNS) was identified as a potent regulator of bone metabolism. In vivo studies by our group have shown that TH interacts with the SNS to regulate bone mass and structure, and that this interaction involves α2 adrenoceptor (α2-AR) signaling. We have also identified the presence of α2A- , α2B- e α2C-AR subtypes in the epiphyseal growth plate (EGP) of mice. In addition, we have found that mice with isolated gene deletion of α2A-AR and α2C-AR (α2A-AR -/- and α2C- AR -/- ) show a disorganized EGP, shorter long bones and a delay in endochondral ossification (EO). In vivo studies revealed that the EGP of α2A-AR -/- and α2C-AR -/- animals respond differently (than those of wild-type animals), to TH excess and deficiency, which strongly suggests that TH also interacts with the SNS to regulate bone growth and development. Through a long bone organ culture system, the present study had the goal of investigating if TH interacts with the SNS directly in the skeleton, to regulate the longitudinal bone growth and if α2-AR is involved in this process. Therefore, we evaluated the linear bone growth of tibias derived from 15.5-day-old WT and α2C-AR -/- mouse embryos (E15.5) for 6 days. We have seen that the KO tibias showed a lower longitudinal growth when compared to WT tibias, and that treatment with 10-8 M triiodothyronine (T3) significantly decreased the longitudinal growth of the WT tibias, which was not seen in the KO tibias. We found that the treatment with UK 14.304 (UK), a non-selective α2-agonist, induced the longitudinal growth only of the KO tibias. The expression of genes related to the terminal differentiation of chondrocytes (Col X, IGF-1, Wnt-4 and Runx2) was shown to be increased in the KO tibias (when compared to the expression in WT tibias). Treatment with T3, as expected, stimulated the expression of these genes in WT tibias, but decreased the expression in KO samples, highlighting the importance of these receptors in the modulation of T3 actions. We observed that the local ? 2 -AR activation by UK blocked the expression of these chondrocyte differentiation- related genes, in addition to blocking the positive effects of T3 (UK + T3) in the expression of these genes. These findings show that α2 adrenoceptors act directly in the skeleton, to control the terminal differentiation of chondrocytes and, therefore, the longitudinal bone growth, in addition to allow an interaction with the TH signaling pathway to control these processes. In conclusion, this study shows that TH interacts with the SNS, locally in the skeleton, via α2 adrenergic signaling, to modulate the longitudinal bone growth.

O diabetes mellitus (DM) é a endocrinopatia mais prevalente no planeta. Na vigência de DM e de obesidade a inflamação subclínica, em decorrência do aumento de infiltrado de células inflamatórias e aumentada expressão de citocinas inflamatórias em tecidos essenciais no controle da homeostase glicêmica, tem importante participação no desenvolvimento da resistência à insulina. Em estados de hipo ou hipertireoidismo ocorre redução captação de glicose em resposta à insulina no tecido adiposo e muscular esquelético, o que reforça a importância da integridade da função tireoidiana para o controle glicêmico. Recentemente, diversos estudos vem apontando uma correlação positiva entre DM e disfunções tireoidianas. Neste contexto, a hipótese do presente estudo foi de que o tratamento com triiodotironina (T3) promove efeito benéfico na sensibilidade à insulina em modelos experimentais de DM ou obesidade, por modulação da expressão de citocinas inflamatórias. Para testar nossa hipótese, ratos DM tipo 1 DM1 (indução por aloxana) ou obesos (indução por dieta de cafeteria) foram tratados com T3 por 4 semanas. Os ratos DM1 apresentaram hipotireoidismo primário e aumento de infiltrado de células inflamatórias no tecido adiposo periepididimal (TAP) e da expressão de fator de necrose tumoral α (TNF-α) e interleucina-6 (IL-6) no TAP e no músculo sóleo. O tratamento com T3 promoveu redução da glicemia, aumento da sensibilidade à insulina, e redução da expressão de TNF-α e IL-6 no TAP e no músculo sóleo. Os animais obesos apresentaram hipertireoidismo subclínico, aumento da glicemia, e redução da sensibilidade à insulina. O tratamento com T3 nesse modelo experimental promoveu aumento da sensibilidade à insulina e da tolerância à glicose e redução da expressão de TNF-α e IL-6 no TAP e no músculo sóleo. Apesar dos efeitos colaterais do T3 na função cardíaca, não foi observado nenhuma alteração na pressão arterial dos animais DM1 tratados com T3. A partir do presente estudo, pode-se concluir que o tratamento com T3 promove melhora da sensibilidade à insulina em ratos DM1 e obesos, mecanismo que envolve a redução da expressão de citocinas inflamatórias no tecido adiposo e muscular esquelético. / Diabetes meliitus (DM) is the endocrinopathy most prevalent around the world. The subclinical inflammation in DM and obesity has important role on insulin resistance development and is caused in consequence of increased inflammatory cells infiltration and expression of inflammatory cytokines in tissues which are essential to glucose homeostasis. Hipo or hiperthyroidism states there is decreased insulin-induced glucose uptake in adipose tissue and skeletal muscle, which reinforces the importance of thyroid function integrity to glycemia control. Lately, several studies have pointed positive relationship between DM and thyroid dysfunctions. In this context, we hypothesis of the present study was that triiodothyronine (T3) could promotes benefitial effects on insulin sensitivity by altering expression of inflammatory cytokines in experimental models of DM or obesity. In order to this hypothesis, type 1 DM - DM1 (alloxan induced) or obese (cafeteria diet induced) rats were treated with T3 during 4-week period. The DM1 rats presented primary hypothyroidism and increased inflammatory cells infiltration and expression of inflammatory tumoral necrosis factor (TNF-α) and interleukin-6 (IL-6) on epididymal adipose tissue (EAT) and soleus skeletal muscle. The treatment with T3 promoted reduction of glycemia, improvement of insulin sensitivity and decrease on expression of TNF-α and IL-6 in EAT and soleus skeletal muscle. The obese rats presented subclinical hyperthyroidism, increased glycemia and reduction of insulin sensitivity. The treatment with T3 in this experimental model promoted increase on insulin sensitivity and glucose tolerance, furthermore, reduction in expression of TNF-α and IL-6 in EAT and soleus skeletal muscle. Even though, side effects of T3 on cardiac function, it was not observed any alteration in arterial pressure of DM1 rats treated with T3. Taken together the results of the present study, it could be conclude that treatment with T3 promotes improvement on insulin sensitivity in DM1 and obese rats, mechanism which involves reduction in expression of inflammatory cytokines on adipose tissue and skeletal muscle.

Sabe-se que o hormônio tireoideano (HT) regula o desenvolvimento e crescimento dos ossos. No estudo, investigamos se o HT interage com o sistema nervoso simpático (SNS) controlando o crescimento longitudinal ósseo (CLO), e se essa possível interação depende do α2A-AR. Para tanto, avaliamos o efeito de 30 dias de hipotireoidismo (HIPO) e hipertireoidismo (HIPER) e α2A-AR-/- de 21 dias de idade. Vimos que os animais α2A-AR-/- apresentam menor comprimento no fêmur, tíbia, rádio, úmero e L4 quando comparados aos animais Selv. Como esperado, o HIPO e HIPER prejudicaram o CLO desses ossos nos camundongos Selv, entretanto, o efeito do HIPO foi mais deletério. A morfologia da LE distal do fêmur mostrou que os animais α2A-AR-/- eutireóideos (EUT) apresentam desorganização de zonas e alterações no número de condrócitos sendo o Hipotireoidismo o tratamento mais deletérios. Dados desse estudos sugerem que as vias PTHrP/Ihh e IGF-1/IGF-1R possam ser vias de convergência do SNS e HT na regulação da morfofisiologia da LE, envolvendo o α2A-AR. Observou-se que os animais α2A-AR-/- apresentam alterações no osso trabecular com potencialização com o hipotireoismo. Além disso, os animais α2A-AR-/- apresentam alterações a conectividade trabecular. Esses achados sugerem que o SNS e interage o HT dependente do α2A-AR. / It is known that the thyroid hormone (TH) regulates the development and growth of bones. In the study, we investigated whether the HT interacts with the sympathetic nervous system (SNS) controlling bone longitudinal growth (CLO), and if this possible interaction depends on α2A-AR -/-. Therefore, we evaluated the effect of 30 days of hypothyroidism (HYPO) and hyperthyroidism (HYPER) and α2A-AR -/- 21 days old. We have seen that α2A-AR -/- animals have shorter femur, tibia, radius, humerus and L4 compared to Selv animals. As expected, the HYPO and HYPER damaged the CLO of these bones in Selv mice, however, the effect of HYPO was more deleterious. The morphology of the distal femur LE showed that α2A-AR -/- animals euthyroid (EUT) have clutter zones and changes in the number of chondrocytes and Hypothyroidism most harmful treatment. Data from this study suggest that PTHrP pathways / Ihh and IGF-1 / IGF-1R can be SNS convergence and HT pathways in the regulation of the CO morphophysiology involving the α2A-AR. It was observed that α2A-AR -/- animals show changes in trabecular bone with augmentation with the hipotireoismo. Furthermore, α2A-AR-/- animals show changes trabecular connectivity. These findings suggest that the SNS and interacts the HT dependent α2A-AR.

O objetivo deste estudo foi investigar os efeitos do T3 e do seu análogo GC-24, agonista TR<font face=\"symbol\">b seletivo, na proteólise muscular mediada pela via ubiquitina-proteassoma. Avaliamos o efeito do T3 e GC-24 no trofismo radial de fibras musculares, no nível de ubiquitinação e na expressão de genes envolvidos na via ubiquitina-proteassoma. Para tanto foram utilizados, ratos Wistar divididos em 4 grupos (Controle, 12 horas, 1 e 7 dias) e tratados com T3 e GC-24. Determinou-se a área de secção transversa dos cortes histológicos através do programa \"Image Pro-Plus\". O nível de ubiquitinação foi determinado através de Western Blot para proteína ubiquitinada e a expressão gênica por PCR em Tempo real. T3 e GC-24 promoveram redução do diâmetro das fibras musculares e aumentaram o nível de proteínas ubiquitinadas em ambos os músculos. Com relação à expressão gênica, T3 e GC-24 modularam a expressão dos genes analisados de maneira diferenciada, demonstrando que GC-24 é capaz de modular genes pouco ou não responsivos ao T3. / Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms a/pha (TRa) and beta (TRI3). Abnormalities in skeletal muscle function have been associated with increased leveis of T3, which is a major sarcopenia (Ioss of sarcomeres). Although the phenomenon of sarcopenia induced by T3 has been widely reported, little is known about the molecular mechanisms invo/ved in proteolysis induced by T3. In this study we have investigated the effects of T3 and GC-24, a novel synthetic TRI3¬selective compound, on the ubiquitin proteasome pathway. We analyzed the effect of T3 and GC-24 on the radial trophism, ubiquitination leveis and gene expression of the ubiquitin-proteasome pathway, which are important regulators of muscle proteolysis in the skeletal muscle. We have addressed the ubiquitin ligases (Atrogin¬1, MuRF-1 and E3a) and the deubiquitinating enzymes (UBP45, UBP69 and USP28). Wistar male rats (170-200g) were divided in 4 groups (Control, 12, 1 and 7 days). Rats received T3 (30l-\'g/100g) and GC-24 (16 I-\'g/1 OOg). After decapitation, EDL and soleus muscles were removed for histological ana/ysis, protein expression and gene expression. Cross sectional area was determined in histological sections through the software \"Image-Pro Plus. The ubiquitination leveis was determined by Western Blot and gene expression determined by Real Time PCR analysis. T3 and GC-24 reduced the diameter of the muscle fibers vs control group. Both T3 and GC-24 incresed the ubiquitination leveis, in the soleus and EDL. Regarding gene expression analysis, T3 and GC-24 modulate the gene expression in a differential manner. In the soleus, T3 increased Atrogin-1 and E3 alpha gene expression, while did not alter Murf-1 gene expression. On the other hand, in EDL Atrogin-1 gene expression is not altered, while E3 alpha and Murf-1 are elevated by T3. In the soleus and EDL deubiquitinating gene expression is mostly not altered, exception made for UBP 45, which is reduced by T3 in soleus muscle. GC-24, increased gene expression of E3a and MuRF-1 in the soleus, while did not alter Atrogin-1 gene expression. However, in EDL muscle, GC-24 increased Atrogin-1 and E3a mRNA, while did not alter MuRF-1. Finally, GC-24 decreased UBP 45 gene expression in EDL muscle and USP 28 gene expression was robustly elevated by GC-24 in both muscles analyzed. This data shows that GC-24 is able to strongly modulate genes that are less responsive or even unresponsive to T3, pointing that the GC-24-TRb complex might trans-activate differently target genes. However, both T3 and GC-24 are able to modulate the muscle proteolysis.

Orientador: Célia Regina Nogueira / Resumo: O câncer de mama (CM) é o primeiro em taxa de mortalidade em mulheres no mundo e o segundo mais frequente entre as mulheres no Brasil. Os fatores de risco estão relacionados com a idade, fatores ambientais, comportamentais, genéticos/hereditários, história reprodutiva e hormonal, bem como os hormônios tireoidianos (HT) que têm sido propostos por influenciar o desenvolvimento de CM. A superexpressão do fator induzido por hipóxia 1 subunidade alfa (HIF1A) e o fator transformador de crescimento alfa (TGFA) está positivamente relacionada com a agressividade do tumor e com a progressão maligna em neoplasias humanas, porém não foi avaliada a participação do hormônio triiodotironina (T3) na expressão desses genes em vias extranucleares MAPK/ERK e integrina αvβ3. No CM, a desregulação na expressão de microRNAs (miRNA) têm sido detectada em casos metastáticos e de pior prognóstico, sugerindo funções importantes na oncogênese mamária e na progressão do câncer. Nosso objetivo foi verificar o envolvimento do T3 no controle pós-transcricional da expressão de mRNA HIF1A e TGFA e avaliar se os miRNAs envolvidos no CM, let-7a-5p, miR-200a-3p e miR-335-5p, são modulados por esse hormônio em células de adenocarcinoma de mama MCF7. A linhagem celular de adenocarcinoma de mama MCF7 foi submetida ao tratamento com T3 na dose suprafisiológica (10-8M) por 1 hora, na presença ou ausência de drogas específicas para o bloqueio de vias de sinalização intracelulares para abordagem de ações extranucleare... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

It has been shown that thyroid hormones are important in development and growth in birds and further that thyrotropin (TSH) signaling regulated photoinduced seasonal reproduction. In addition to controlling the development of certain physiological traits, TSH can affect a wide range of phenotypes related to domestication such as behaviour, growth rate, more frequent reproductive cycle’s, pigmentation and also behaviour. Studies indicate that thyroid hormone physiology could potentially be responsible for differences in individual stress response as well as differences in social dominance. This project investigated behaviours expressed in the different genotypes on the Thyroid stimulating hormone receptor (TSHR) gene in chickens. Standard test such as Fear of human, Aerial predator, Tonic immobility and Social hierarchy were carried out with White leghorn (WL) as a domesticated species and Red Junglefowl (Rjf) as their wild counterpart; these were considered to be the control group. There was no significant result on genotype effect for the TSHR animals observed in those variables tested. The TSHR mutants were expected to mirror the White leghorn behavioural response and the TSHR wildtype the behaviour of Rjf. This was however not confirmed. There were a significant interaction between genotype effect and sex effect for TSHR for stand alert in the Aerial predator test which mirrored the results seen in the control groups. The male wildtype followed the male Rjf pattern however the mutant did not mirror the WL male. This study would benefit from more individuals to be tested, for stronger statistical results, plus also to have all genotypes represented to fully investigate the affect the TSHR mutation have on domesticated chickens and potentially the domestication process in a range of species. / TSHR

<p>Polybrominated diphenyl ethers (PBDEs) are a class of flame retardant chemicals that were added to furniture foam, electronics, plastics, and some textiles to reduce their flammability. While PBDEs have been phased out from use in current products, huge reservoirs of products containing PBDEs still exist. It is likely exposure to PBDEs will continue as older products are discarded and recycled. PBDEs are ubiquitous contaminants in indoor and outdoor environments due to their widespread use in many products and their ability to migrate out of treated materials.</p><p> Major health effect concerns from PBDE exposure identified in laboratory studies include neurotoxicity, reproductive/developmental toxicity, and thyroid disruption. Importantly, mammals metabolize PBDEs into the hydroxylated polybrominated diphenyl ethers (OH-BDEs), which are structurally similar to endogenous thyroid hormones. Thyroid hormones are essential for metabolic processes, growth, and development, particularly brain development. Multiple studies have demonstrated enhanced potency of OH-BDEs relative to the parent PBDE chemicals, particularly for neurodevelopmental processes. Additionally, in fish species, thyroid hormones are essential for transitioning between larval, juvenile, and adult life stages. Therefore, studying the effects of both PBDEs and OH-BDEs during sensitive developmental life stages (i.e. larval and juvenile development) is warranted. The hypothesis of this thesis research is that PBDE metabolites interfere with thyroid hormone signaling (through interacting with thyroid receptor and deiodinase enzymes) which may result in decreased growth, morphological deficits, and altered neurodevelopment. The objectives of this research project were to evaluate the toxicity of PBDE metabolites and mixtures of PBDEs/OH-BDEs on larval and juvenile zebrafish development, examining both potential modes of action as well as functional consequences of exposure in developing animals. </p><p>In the first aim of this thesis research, structural relationships were examined between eleven different halogenated phenolic compounds (OH-BDEs, OH-PCBs, halogenated phenols, and TBBPA) to test developmental toxicity in zebrafish from 0-6 days post fertilization (dpf). In addition, follow up studies were performed with the most toxic compound, 6-hydroxy- 2,2’,4,4’-tetrabromodiphenyl ether (6-OH-BDE-47), to examine effects on TH-mediated morphological development and to better understand its mechanism of action in zebrafish. Thyroid disrupting agents including propylthiouracil, iopanoic acid, and native thyroid hormones were also used as positive controls for morphologic studies. Exposures to 6-OH-BDE-47 (10 nM to 100 nM) during development resulted in severe delays, similar to exposures from the T3 and thyroid disrupting agents. Lower jaw deformities and craniofacial cartilage malformations were also observed following exposure to 6-OH-BDE-47 at doses greater than 50 nM. Of interest, these developmental delays were rescued by overexpression of TRβ mRNA during the exposure period. These data indicate that OH-BDEs can adversely affect early life development of zebrafish and suggest they may be impacting thyroid hormone regulation in vivo through downregulation of the thyroid hormone receptor. </p><p>In the second aim of this dissertation research, neurobehavioral performance was monitored in larval and juvenile fish following a developmental exposure to 6-OH-BDE-47. 6-OH-BDE-47 has been identified as a neurotoxicant in previous cell based assays, and was identified as overtly toxic to zebrafish larvae in Aim 1 of this research. Developmental exposures (0-6 dpf) to 6-OH-BDE-47 resulted in decreased larval swimming activity at 6 dpf, with persisting impacts on behavior at 45 dpf. Young adult fish, when tested at 45 dpf, exhibited increased fear/anxiety response in the novel tank diving task and hyperactivity in a test of sensorimotor habituation. These data indicate that exposures to PBDE flame retardants and their metabolites during critical developmental windows can alter long term cognitive responses more than a month after the exposure has ceased.</p><p>Finally, for the third aim of this dissertation research, zebrafish undergoing larval-juvenile metamorphosis were exposed to a mixture of PBDEs (30-600 µg/L DE-71) and OH-BDEs (1-300 nM) from 9-23 dpf. Metamorphosis is a unique developmental period in fishes which is partially mediated by thyroid hormones. Juvenile animals, like larval animals, represent a sensitive and unique subpopulation of animals. At the end of the exposure period (23 dpf), a subset of fish were reared in clean water until 45 dpf for neurobehavioral testing. Fish samples were collected at 3 time points throughout the experiments, Days 12, 23, and 45. Tissue accumulation of test chemicals was monitored, and juvenile fish treated with the High Mixture were found to accumulate over 100 µg/g ww ∑PentaBDEs. The highest mixture treatment was found to be acutely toxic to zebrafish juveniles, resulting in >85% mortality within 14 days of exposure. Fish treated with 30 nM 6-OH-BDE-47 or the lower mixture exhibited reduced morphology scores relating to fin, pigmentation, and swim bladder maturation. In addition, reduced skeletal ossification and caudal area was observed at earlier time points with treatment to 6-OH-BDE-47. These alterations were accompanied by increases in chondrogenic gene expression, declines in osteogenic gene expression, and increases in thyroid receptor expression. Approximately 3.5 weeks after the exposure period, juvenile fish were tested on neurobehavioral tasks of novel tank exploration and sensorimotor habituation, however, no significant treatment related effects on task performance were observed. Collectively, these data suggested that the larval/juvenile development stage is a sensitive developmental window which can be adversely impacted by PBDE/OH-BDE exposure.</p> / Dissertation